Metabolite Comparison between Spleen-Deficiency and Healthy Children

Objective From the perspective of metabolomics, this study compares the metabolomics characteristics of feces and urine between children with spleen-deficiency and healthy children to explain the scientific connotation of children with spleen-deficiency susceptibility to digestive system diseases from the metabolic level and provide a scientific basis for further research. Methods This study included 20 children with spleen-deficiencies and 17 healthy children. Children's symptom scores, height, and weight were recorded in groups, and feces and urine samples were collected. The samples were detected using ultrahigh-performance liquid chromatography-mass spectrometry. The data were analyzed using multivariate statistical analysis such as principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA). Related differential metabolites were identified through database comparisons between two groups based on the MS and KEGG. Results Compared to healthy children, the metabolites glucuronic acid, xanthine, and indole-3-acetaldehyde tend to be reduced in children with spleen-deficiency. Moreover, these children showed an increase in metabolites such as quinic acid, adenine, 4-methyl-5-thiazole-ethanol, 3-formyl indole, and 5-hydroxy indole-3-acetic acid. The condition affected many of the critical metabolic pathways, including the metabolism of tryptophan, cysteine, methionine, and pentose phosphate. Conclusion The children with spleen-deficiency had disorders at the metabolic level, which might be due to factors such as diet, personal preferences, and genes, leading to various symptoms, making spleen-deficiency children more prone to suffer from digestive diseases than healthy children. The results set a basis for the research on children's TCM constitution, which can be a reference to further studies to deal with the spleen-deficiency.


Introduction
People have diferent characteristics of sufering from the same disease due to various classifcations such as gender, race, and region [1][2][3][4]. For instance [3], researchers found that MSspecifc mortality trends demonstrate distinctive disparities by race/ethnicity and age. In traditional Chinese medicine (TCM) [5], there is also a kind of classifcation approach called constitution classifcation, which has similar functions to those classifcations mentioned above but only diferent names. Dong's team had attempted to help the theory of the TCM constitution move towards internationalization in the translation of the academic term, and names will not afect the connotation of the classifcation methods [6].
A TCM constitution refers to the comprehensive and relatively stable inherent characteristics of the human body in terms of morphological structure, physiological function, and psychological state that are formed based on the innate and acquired endowment [7]. Te TCM constitution is the internal basis of disease occurrence, development, transformation, and prognosis. TCM doctors can diagnose a person's constitution by identifying patterns and then infer their potential disease spectrum by analyzing the constitution, even if the patient is not "sick" as per modern diagnostic techniques [8].
People with the exact constitution have similar susceptibilities to certain diseases. Liang's team [4] preliminarily revealed the correlation between constitution and diseases in 1639 studies in 2020. A report showed the relationship between allergic rhinitis and constitution in 2022 [9]. And in 2021, a cross-sectional study demonstrated the association of constitution and female patients with systemic lupus erythematosus [10]. More and more research on the TCM constitution show that targeted treatments for people with diferent constitution types will help reduce the risk of sufering from certain diseases.
Te constitution analyses also apply to children even if there is no targeted standard of the TCM constitution for them [11,12]. However, there is not much international research about children on the TCM constitution. Since children with spleen-defciency account for much of children's constitution classifcation, we chose this constitution as our objective [13]. Tese children normally have symptoms like emaciation, listlessness, fatigue, sweating, poor appetite, indigestion, muscle weakness, irregular stool, or even persistent diarrhea. Teir growth and development are slower than those of healthy children (called balanced constitution in TCM), which easily afects the function of the digestive and other systems, making spleen-defciency children more susceptible to digestive diseases [14][15][16][17]. For instance, spleen-defciency may result in phlegm dampness, which can easily cause lung diseases such as cough and asthma [18]. Childhood body physique is the foundation of lifelong health, and the subhealth status of children with spleendefciency is associated with numerous families. Children's health is closely related to the socioeconomic status of the country and the nation; it is an urgent concern of parents and clinicians to ensure children's healthy growth and fnd a way to improve their spleen-defciency constitution. Terefore, our research aimed to investigate the distinct indicators between spleendefciency and healthy children.
Metabolomics can efectively reveal the essence of an organism's metabolism and accurately refect the biological system's state by exploring micromolecular metabolites and metabolic pathway products, which have been used in TCM constitution research [19][20][21]. In 2018, Chen's team [22] found that diferentially expressed miRNAs may partly explain the TCM constitution's distinct characteristics, showing an excellent research example. And there is no report on the microscopic characterization of spleen-defciency in children, either domestically or internationally.
Tus, this study aims to reveal the characteristics of small molecular metabolites and metabolic pathway products in children with spleen-defciency and provide new ideas for improving the physique of such children and preventing related diseases.

Sources and Grouping of Cases.
Children in the spleendefciency group were enrolled from October 2019 to December 2020 in the Pediatric Tuina Department, the Pediatric Outpatient Department of the First Afliated Hospital of Hunan University of Traditional Chinese Medicine, and the Pingdi Kindergarten of Yueyang City, aged between one and six years. Te healthy group was the openly recruited children from Pingdi Kindergarten in Yueyang City who were in good condition per the physical examination. According to the TCM identifcation standard, 20 children with spleen-defciencies and 17 healthy children were included. Te process of the experiment and the symptom list of 20 spleen-defciency children are as follows (see Figure 1 and Table 1). (Te entire name is hidden in Table 1 for privacy concerns).

Ethics Committee Approval. Te Medical Research Ethics
Committee of the Xiangxi Hospital of Chinese Medicine of the Tujia and Miao Ethnic Group provided the approval. All the participants' parents signed an informed consent form, and all work was conducted by the Declaration of Helsinki (1964) during the process (Protocol Registry Number: 2019-No. 08)

Sample Collection and Storage
2.4.1. Feces. About 30 mg of fresh feces from the participants was collected in the morning, and the feces were placed in a cryopreservation tube. Te sample was then brought to the laboratory at an ordinary temperature within 1 hour and stored in a refrigerator at −80°C.

Urine.
Te midstream urine of the participants was collected in the morning using a disposable urine collection cup, and about 10 mL of urine was poured into a cryopreservation tube. Te sample was then brought to the laboratory at an ordinary temperature within 1 hour and stored in a refrigerator at −80°C.

Sample Preparation
2.5.1. Feces. First, the samples were thawed at 4°C and swirled for 30 min. Ten, 10 mg of the samples were weighed, 1000 μL of 1% formic acid/acetonitrile mixture was added after an ice bath, and 20 μL of 1000 μg/mL internal standard was added, swirled for 1 min, and allowed to stand for 10 min.
Te sample was centrifuged at 4°C, 13000 rpm for 5 min, and 800 μL of the supernatant was collected in a centrifuge tube and fushed with nitrogen. Ten, 100 μL of acetonitrile/ water/formic acid (80 : 19 : 1, v/v/v) was added to re-dissolve the sample. Te sample was centrifuged again under the same conditions as described above. Finally, 70 μL of the supernatant was collected and tested.

Urine.
First, the samples were thawed at a temperature of 4°C. Te samples were centrifuged at 4°C, 13000 rpm for 5 min. All supernatants were fltered with a 0.22 μm aqueous phase fltration membrane. After that, 80 μL was taken, and 20 μL of 1000 μL g/mL internal standard was added, swirled for 30 s and tested.

Liquid Chromatography-Mass Spectrometry (LC-MS/MS) Metabolomics
Test. Te positive ion mode mobile phase was an aqueous solution containing 0.1% formic acid (liquid A) and 100% methanol containing 0.1% formic acid (liquid B). In contrast, the negative ion mode mobile phase was an aqueous solution containing 10 mM ammonia formate (liquid A) and 95% methanol containing 10 mM ammonia formate (liquid B). Te details are presented in Tables 2  and 3 [23] and the related description of children's biased constitution in Pediatric Massage [24] (the textbook of the 13 th Five-Year Plan of National Higher Education edited by Professor Liao Pindong in 2016) were used as TCM spleen-defciency identifcation standard. Te main indexes included were (1) diarrhea or incomplete defecation, (2) sallow yellow or wan complexion, (3) poor appetite, (4) emaciation or pufness, (5) light or fat tongue with teeth marks and greasy coating. Secondary indexes were (1) dark around the eyes, (2) spontaneous sweating or hyperhidrosis, (3) fnger-licking and salivation, (4) mild anemia, (5) mild edema, (6) opening eyes while sleeping, (7) abdominal discomfort, (8) limb weakness and fatigue, and (9) faint fnger venules and a weak pulse.
If three main or two main indexes combined with two secondary indexes were met (at least) and the symptoms persisted for over three months, the condition was classifed as spleen-defciency.
Balanced constitution [25]: A balanced constitution was defned as follows: normally developing, well-proportioned, muscular, energetic, lively, fexible, quick-reacting children with peaceful sleep, warm limbs, a moist skin, dark hair, moist complexion, vivid eyes, even breathing, loud crying, a strong voice, moderate diet, normal defecation, a pale red throat, a pale red tongue, and a thin and white tongue coating. Children with a balanced constitution can tolerate frequent climate change more efciently and have fewer illnesses and allergies than other children. In this essay, we use "healthy children" as pronouns.

Inclusion Criteria.
Children meeting the following criteria are as follows: (1) meeting the identifcation standard mentioned above for children's spleen-defciency constitution and healthy children; (2) age range: 1--6 years, regardless of gender; and (3) parents of children ready to participate and sign the informed consent form voluntarily.

Exclusion Criteria.
Te exclusion criteria are as follows: (1) skin with burns, scratches, scabies, trauma, fractures, and bone dislocation; (2) acute infectious diseases such as osteomyelitis, cellulitis, erysipelas, and bone tuberculosis; (3) bacillary dysentery, amoebic dysentery, and cholera; (4) severe systemic diseases such as a serious malignant tumor, heart disease, psychosis, and liver and kidney diseases; and (5) use of hormone drugs or other traditional Chinese and western medicines or immunomodulators in the last three months.  Evidence-Based Complementary and Alternative Medicine . Te data were preprocessed for peak identifcation, automatic integration, and retention time correction using the MS-DIAL software platform. A visual matrix containing metabolite number, retention time, mass-to-charge ratio, metabolite name, ion mode, and peak area were obtained. Subsequently, the feature number of samples was screened to obtain a result containing peak quantitative results (within sample names, ms1 and ms2) and the visual matrix (including the relative quantifcation of peak areas). Te substances were characterized according to the data of ms2, and a variety of metabolites were identifed by comparing them with the MS Bank database. Te comparison of the database is qualitative and then relatively quantitative by peak area, and the comparison between samples is presented by data analysis. Te qualitative indicator is that substances with a score above 0.7 will be displayed in the qualitative table.
Ten the relationship between multiple and individual metabolic pathways was analyzed using graphical functions. Principal component analysis (PCA) and partial least squares analysis (PLS-DA) were used to acquire the corresponding scores and loading plots, and the results were analyzed and compared. VIP values were extracted from the partial least-square discriminant analysis score chart. Generally, variables with a VIP > 1 have signifcant diferences. Using the signifcant diferences to obtain the following results, such as heatmap analysis and metabolic enrichment analyses.    Secondly, use lowess to process the fuctuating data to achieve data ftting and normalization. Te data obtained were saved in an Excel fle for analysis and processing.

Other Data Analysis.
Other data were collected and sorted in Excel, compared, and analyzed using SPSS 21.0. Statistical signifcance was set at P < 0.05. Te measurement data were expressed by "mean standard deviation" (x ± S). When a normal distribution was met, the paired t-test was used. Te rank-sum test was used when the normal distribution was not satisfed, and the chi-square test was employed to analyze the counting data.

Basic Information Comparison.
Te basic information comparison between the healthy and spleen-defciency group can be seen in Table 4.

Specimen Test Results of Feces and Urine.
Tere are some legends in the upper-right corner of Figures 2-6, "A" or "a" represents the feces or urine results of the spleen-defciency group, and "C" or "c" represents the feces or urine results of the healthy group.

Ion Map.
Te total ion fow graphs of all QC samples were superimposed. Te spectrogram overlapped well, and the retention time and peak signal intensity fuctuated little, indicating that the instrument was in good condition during the whole sample analysis process, which was suitable for the detection and analysis of the project. According to the LC-MS/MS condition in the research methods, sample data were collected, and the total ion current was determined, as displayed in Figure 2. Te chromatogram indicated diferences in the number and height of chromatographic peaks in the feces of each group in positive and negative ion modes. Tese indicated substantial metabolites diferences in feces and urine between the spleen-defciency and healthy groups, which can be further analyzed and compared.

PCA Score Chart.
PCA is an unsupervised learning model which plays an important role in data dimensionality reduction. Tere were 643 metabolites with signifcance, 286 for feces and 357 for urine; they were used to generate the PCA and PLS-DA models. It is a k-dimensional feature reconstructed based on the original n-dimensional part. Based on the ion maps, the PCA score chart could be drawn.
First, the data was normalized and transformed by the Pareto scaling method. Ten a set of mutually orthogonal coordinate axes in sequence from the original data square was found, and the choice of new coordinate axes was closely related to the data itself. Among them, the frst new coordinate axis was the direction with the most signifcant 0  1  2  3  4  5  6  7  8  9  10  11  12  13  14  15  16  17  Retention Time (min)  0  1  2  3  4  5  6  7  8  9  10  11  12  13  14  15  16   Evidence-Based Complementary and Alternative Medicine variance in the original data, the second new coordinate axis was the plane orthogonal to the frst coordinate axis, and the third axis was the plane orthogonal to the frst and second axes. And so on, we got n coordinate axes. We found that in the new coordinate axes obtained in this way, most of the variance was contained in the frst k coordinate axes, and the variance in the last coordinate axes was almost zero. So, we ignored the rest of the coordinate axes and only kept the frst k coordinate axes with the most signifcant variance. Tis procedure was equivalent to retaining only the dimension features that contain most variance, and ignoring the feature dimensions that contain almost zero variance to reduce the dimension of data features. As depicted in Figure 3, each group of samples' metabolic profle was clear. Te pink parts stand for the spleendefciency group and the green ones for the healthy group.   Evidence-Based Complementary and Alternative Medicine Te ellipse represents the 95% confdence interval of a group of samples. Te fgure showed partial conglomerations of the two groups of models in the positive and negative ion modes with a pretty reappearance. A specifc separation between the two sample groups indicates a diference in sample metabolism. Te result lays a foundation for understanding the diferential metabolic characteristics of the two groups and then comparing and analyzing PLS-DA results between them.

PLS-DA Score Chart.
Te PLS-DA is a statistical method related to the PCA. After the reducing of the dimension of data features, the PLS-DA model was established. It projected the predicted and observed variables into a new space by way of projection to establish regression analysis between group (Y) and metabolite (X). A linear regression model was found during the process, which was used to fnd the diference between groups and get diferential metabolites. Ten several kinds of fgures were obtained.

Evidence-Based Complementary and Alternative Medicine
In the PLS-DA score chart (see Figure 4), the pink parts stand for the spleen-defciency group and the green ones for the healthy group. Te discrete and clustered fgures showed the reproducibility of the samples and the metabolic profle between the sample groups. Tat means models in the same group were similar, while the two groups showed diferent metabolism features. A clear separation of the two groups of samples can be seen in the score chart. Te metabolic profles of feces and urine of children with spleen-defciency and healthy children demonstrated signifcantly separated features.

Cross-Validation of PLS-DA.
Cross-validation (CV) was used to evaluate the quality of the model. CV divided the sample data into two parts: the training set and the verifcation set. Te training set was used to build the model, and the verifcation set was used to test the model. R 2 is the accuracy of the model establishment. Q 2 is the prediction accuracy of the model. Te higher the Q 2 and R 2 are, the higher the model's quality and prediction ability are. Figure 4 is the resulting diagram of the cross-validation of the PLS-DA model. Te abscissa shows the number of components the model selects, and the ordinate shows the values of R 2 , Q 2 , and accuracy. Generally, the group with the highest R 2 and Q 2 values was assigned. As depicted in Figure 5, the model had qualifed prediction ability and accuracy.

VIP Diagram of PLS-DA.
Te VIP diagram of PLS-DA can help people understand the contribution of diferent groups to sample diferentiation and the signifcance of variables. After the model establishing, the contribution of each variable could be observed. VIP values were extracted from the PLS-DA score chart. Generally, variables with a VIP > 1 have signifcant diferences. Te diferential metabolites with most signifcance were selected to form the fgures. As depicted in Figure 6, "A" or "a" represents the feces or urine results of the spleen-defciency group, and "C" or "c" represents the feces or urine results of the healthy group. Te horizontal coordinates in the coordinate system on the left side of the fgure are the VIP values, the vertical coordinates are the diferential metabolites, and the color coding on the right side refects the high and low concentrations of this metabolite in the diferent groups. Te higher the VIP value is, the more signifcant the contribution to sample diferentiation. Te VIP score of metabolites in Figure 6 was more than 1, contributing signifcantly to the diferences in the samples. Te VIP colors and fgures of the groups were diferent, indicating signifcant diferences in the metabolites in feces and urine samples between the two groups.

Heatmap Analysis.
Te VIP diagram-obtained differential metabolites were further visualized using heatmap analysis to provide a more intuitive understanding of the diferences and distribution of data. As shown in Figure 7, "A" or "a" represents the feces or urine results of the spleendefciency group, and "C" or "c" represents the feces or urine results of the healthy group. Te color in each gird stood for various data. Te distribution of metabolites in feces and urine samples varies signifcantly between spleen-defciency and healthy children.

Diferences in Metabolites of Feces and Urine.
According to the metabolites (including positive and negative ions) with VIP > 1 in PLS-DA data results, many metabolites required further screening. Trough P < 0.05 and FD > 1.5 or <0.75, it was found that there were three metabolites in the positive ion mode and fve metabolites in the negative ion mode. Among them, glucuronic acid, xanthine, and indole-3-acetaldehyde indicated a downward trend, whereas quinic acid, adenine, 4-methyl-5thiazole-ethanol, 3-formyl indole, and 5-hydroxy indole-3-acetic acid demonstrated an increasing trend (see  Table 5). Partial diferential metabolites are shown in Table 5; the full contents can be reached by connecting with our team.

Metabolic Pathways of Feces and Urine.
Under the basis of analyzing the diferential metabolites, two sets of data were put into the channel of the model organism together. Te metabolic pathways of diferent metabolites were enriched and analyzed to identify the metabolic pathways with signifcant infuence. Ten the bubble diagram of the metabolic pathway is shown in Figure 8. Each ring in the diagram represents a diferent metabolic pathway produced by the metabolic pathway analysis, and the color of each circle represents the P value. Te redder the color, the smaller the P value, the higher the signifcance, and the larger the corresponding −log (P) value. Te size of the circle indicates the infuence of this metabolic pathway. Te larger the circle, the greater its impact. As depicted in Figure 8, if the ring is closer to the upper-right side of the diagonal line, the metabolic pathway is more likely to be sought.
In the analysis of fecal diferential metabolic pathways, there were signifcant diferences between metabolic pathways such as pantothenate and CoA biosynthesis, alanine, aspartate, and glutamate metabolism, vitamin B6 metabolism, and nicotinate and nicotinamide metabolism (see Figures 8(a)-8(c)).
In the analysis of urine diferential metabolic pathways (see Figures 8(b)-8(d)), metabolic pathways such as alanine, aspartate and glutamate metabolism, nicotinate and nicotinamide metabolism, histidine metabolism, taurine and hypotaurine metabolism, and arginine and proline metabolism exhibited signifcant diferences.

Discussion
Te metabolic pathways coincide in positive and negative ion modes in the spleen-defciency children and the healthy group. According to P < 0.05 and considerable impact values, three metabolic pathways with signifcant diferences were screened: the tryptophan pathway, the cysteine and methionine pathway in positive ion mode, and the pentose phosphate pathway (PPP) in the negative ion mode. Highly connected diferential metabolites such as adenine, xanthine, glucuronic acid, and quinic acid, which were with the metabolism changes in comparing two groups, will also be discussed below.
Adenine and xanthine were in the diferential metabolites lists between the spleendefciency and healthy groups. Adenine [29,30] is a component of nucleic acids and an essential part of coenzymes such as ATP and CoA and can be obtained by the hydrolysis of nucleic acids. Its phosphate can stimulate leukocytosis and is used to prevent and treat leukopenia caused by various reasons. All types of white blood cells have diferent defensive and protective functions, and timely and sufcient white blood cells are benefcial to defense against diseases. Xanthine is a derivative of purine that is frequently used as a mild stimulant and bronchodilator [31], especially for treating asthma symptoms [32]. In our results, adenine and xanthine have been proven to be at abnormal levels, which may lead to many health problems. Both are transferred from purine, mainly from the food we eat. Terefore, we hypothesize that the disorder of adenine and xanthine may be caused by improper eating diet or insufcient intestinal absorption, causing children with spleen-defciency to have low immunity, a sensitive constitution, and are prone to illnesses.
Quinic acid [33,34] is closely related to human intestinal metabolism. Quinic acid contributes to the synthesis of tryptophan and nicotinamide in the gastrointestinal tract [35,36]. Nicotinamide plays a role in the metabolism of sugar and protein and can improve the nutrition of humans and animals. Additionally, quinic acid can improve bile, promote digestion, and reduce fat accumulation [37]. According to the results, quinic acid content was higher in spleen-defciency children's bodily excretions than in healthy ones. Terefore, all these functions mentioned above may be less active inside the spleen-defciency children's bodies, perhaps leading to the disorder of fat and other energy metabolisms in them. Abnormal energy metabolisms can increase the risk of sufering from digestive system diseases and may cause children with this constitution to be depressed, prone to fatigue, pufy, or sluggish. In its free form, quinic acid is broadly abundant in plants and can accumulate in copious amounts in cofee, tea, and certain fruits [33]. Because of the active efux mechanism, the intestinal absorption of quinic acid is generally low [35]. Tus, we inferred that the quinic acid disorder of spleendefciency children might be caused by its lower intake and absorption, which may be related to eating diet and intestinal function.
Tryptophan has three metabolic pathways in the human intestine [38,39]. Approximately 90% of tryptophan is converted into dog urine ammonia, 5% is directly metabolized by intestinal fora, and about 3% is transferred to 5-HT. As the results showed, children with spleen-defciency have functional disorders in tryptophan metabolism, and those transfers may be increased or decreased. And tryptophan plays an essential role in intestinal immune tolerance and maintaining the balance of intestinal fora. Its metabolites also afect numerous intestinal functions such as barrier, peristalsis, digestion and absorption, secretion, and immunity [40]. Simultaneously, several studies have demonstrated a clear connection between tryptophan and oxidative stress [41][42][43][44][45]. When the tryptophan metabolism disorder in spleen-defciency children appears, the producing process of dog urine ammonia, 5-HT may be disturbed, and the oxidative stress may be stimulated severely.
All the changes may lead to frequent intestinal symptoms such as anorexia and diarrhea and afect mental factors like mood and sleep. Tryptophan [46] mainly comes from the body's initial protein or daily food; people can increase the body's tryptophan contents by taking particular food. Terefore, eating habits may afect the tryptophan metabolism in spleen-defciency children, making them susceptible to numerous diseases, especially digestive diseases. According to the results, children with spleen-defciency had cysteine and methionine metabolism disorders, while cysteine and methionine beneft the human body. Cysteine plays a signifcant role in glutathione synthesis, increases oxidative stress in malnourished children, and reduces cysteine levels [47]. Methionine [48] is the raw material for synthesizing protein and cystine. Terefore, it positively afects the animal skeletal muscle development and the growth and development of embryos or ofspring. As a functional essential amino acid in animals, methionine has positive efects on regulating animal physiological functions, antioxidant capacity, immune function, and intestinal health [49]. Methionine can also afect the digestion and absorption functions of the intestinal tract by improving the development of intestinal mucosal structure and boosting the activity of intestinal enzymes [50]. Cysteine content decreases when metabolic disorder happens, afecting children's growth and immunity. Compared with healthy children, the slow growth and poor immune ability of children with spleendefciency may be caused by the metabolic disorder of cysteine and methionine. Previous research [51] also showed that the methionine content in the serum of malnourished children was low, which is consistent with the results of this experiment. Cysteine and methionine mainly come from in vitro food intake. Te diference in the pathway may be caused by the lack of relevant input in the diet of children with spleendefciency, which may be related to family eating habits and personal preferences. Te main characteristics of PPP are the direct decarboxylation and oxidative dehydrogenation of glucose, while glucuronic acid is one of the products [52]. In the experimental results, glucuronic acid levels in spleen-defciency children presented a downtrend, indicating that the function of this approach is insufcient. PPP is a common pathway of sugar catabolism in animals, plants, and microorganisms [53]. Regular operation of PPP can help organisms transform nutrients from the external environment into substances. However, PPP is nearly lacking in the skeletal muscles of animals, while most glucose is decomposed through this pathway in adipose tissue, mammary glands, and the adrenal cortex [54]. In vivo, the pentose phosphate pathway provides energy and various raw materials for anabolism [55]. Tere may be a positive correlation between thin children with spleen-defciency and PPP dysfunction. Tose children have less fat to carry on the process of PPP. Meanwhile, the lack of PPP causes their abnormal energy metabolism and low physical function, which can explain why spleen-defciency children are less energetic and less robust than healthy children. Te source of this metabolic disorder may be related to their poor genes, digestion, and absorption.

Conclusion
Te results showed that the metabolism condition varies between children with spleen-defciency and healthy children. Te children's spleen-defciency constitution might result from disorders in tryptophan metabolism, cysteine and methionine metabolism, and pentose phosphate metabolism. Tese metabolism problems might be due to factors such as diet, personal preferences, and genes, leading to various symptoms. Setting up a specialized life and diet plan or choosing a therapy conducive to improving metabolism may improve this situation. Future researchers can explore more aspects of children with spleendefciency to create drugs and therapies to improve their body condition based on more results. Nevertheless, our study's sample size was small, and the study duration was not long. More studies are required to verify the conclusion of this experiment in the future.

Data Availability
Te data used in this study are available from the corresponding author upon request.

Disclosure
Huan Zhong and Mi Liu are co-corresponding authors.

Conflicts of Interest
Te authors declare that they have no conficts of interest.